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Transfer RNAs are the fundamental adapter molecules of protein synthesis and the most abundant and heterogeneous class of noncoding RNA molecules in cells. The study of tRNA repertoires remains challenging, complicated by the presence of dozens of post transcriptional modifications. Nanopore sequencing is an emerging technology with promise for both tRNA sequencing and the detection of RNA modifications; however, such studies have been limited by the throughput and accuracy of direct RNA sequencing methods. Moreover, detection of the complete set of tRNA modifications by nanopore sequencing remains challenging. Here we show that recent updates to nanopore direct RNA sequencing chemistry (RNA004) combined with our own optimizations to tRNA sequencing protocols and analysis workflows enable high throughput coverage of tRNA molecules and characterization of nanopore signals produced by 43 distinct RNA modifications. We share best practices and protocols for nanopore sequencing of tRNA and further report successful detection of low abundance mitochondrial and viral tRNAs, providing proof of concept for use of nanopore sequencing to study tRNA populations in the context of infection and organelle biology. This work provides a roadmap to guide future efforts towards de novo detection of RNA modifications across multiple organisms using nanopore sequencing.

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Ligation by plant and fungal RNA ligases yields an internal 2'-phosphate group on each RNA ligation product. In budding yeast, this covalent mark occurs at the splice junction of two targets of ligation: intron-containing tRNAs and the messenger RNA HAC1 The repertoire of RNA molecules repaired by RNA ligation has not been explored due to a lack of unbiased approaches for identifying RNA ligation products. Here, we define several unique signals produced by 2'-phosphorylated RNAs during nanopore sequencing. A 2'-phosphate at the splice junction of HAC1 mRNA inhibits 5' → 3' degradation, enabling detection of decay intermediates in yeast RNA repair mutants by nanopore sequencing. During direct RNA sequencing, intact 2'-phosphorylated RNAs on HAC1 and tRNAs produce diagnostic changes in nanopore current properties and base calling features, including stalls produced as the modified RNA translocates through the nanopore motor protein. These approaches enable directed studies to identify novel RNA repair events in other contexts.

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As observers of human behavior, infants are faced with a complex flow of motion in which pauses are rare and only occasionally coincide with boundaries between intentional actions. Two studies investigated whether, despite such complexity, 10- to 11-month-old infants (N = 16 for each study) possess skills for parsing ongoing behavior along boundaries correlated with the initiation and completion of intentions. After being familiarized with digitized sequences of continuous everyday action, infants showed renewed interest in test versions in which motion paused in the midst of an actor's pursuit of intentions (interrupting test videos). In contrast, pauses that suspended motion at intention boundary points (completing test videos) sparked no such renewed interest on infants' part. Moreover, basic salience differences between the two types of test videos were not the source of infants' increased interest when intentions were interrupted (Study 2). These findings demonstrate that infants readily detect disruptions of the structure inherent in intentional action, and hence parse ongoing behavior with respect to such structure. Such parsing skill is likely a prerequisite to the development of genuine intentional understanding.

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During a routine analysis of disulfide-linked synthetic heterodipeptides by matrix-assisted laser desorption ionization (MALDI) mass spectrometry with linear detection we observed not only the expected mass of the dipeptide, but also the mass of the individual constituent monomer peptides. This was surprising because the peptide was purified as an intact dipeptide and no overt attempt was made to reduce the disulfide linkage before mass analysis. In contrast, analysis of the same sample by electrospray ionization mass spectrometry gave the mass of the dipeptide only. To investigate this further, two additional model heterodipeptides were prepared and all three were used to systematically study several matrix-assisted laser desorption ionization mass spectrometry parameters. These parameters were three different matrices (alpha-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, and sinapinic acid), both positive and negative modes of detection, and varying the acceleration voltage from 5 to 20 kV. Except for the sinapinic acid matrix where poor-quality spectra were obtained, all three model heterodipeptides fragmented under the tested conditions in a manner consistent with the cleavage of disulfide bonds, although the absolute level was sample dependent. The precise mechanism of disulfide cleavage during analysis is unknown, but the cleavage we observed appears to originate during the initial ionization event. Because the MALDI process involves irradiating samples with a laser, the fragmentation of disulfide-linked peptides that we observe bears some resemblance to light-induced homolytic cleavage of aqueous solutions of the amino acid cystine, although other mechanisms for fragmentation are also possible.(ABSTRACT TRUNCATED AT 250 WORDS)

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The link between psychosocial factors and various health problems may be partially explained by the relationships of these factors to healthy behaviours. The relationships of anger experience, anger expression, hostility and hardiness to healthy behaviours was investigated in 97 healthy women. In multiple regression analysis, 25% of the variance in healthy behaviour was accounted for by hostility (12.5%), education (added 7.5%), and the combination of high anger experience/high anger-out (added an additional 5%). This was especially significant in view of the significant relationship of hostility to both anger experience and anger out. Although hardiness showed a significant positive correlation (r = 0.23 P less than 0.05) with healthy behaviours, it did not enter the multiple regression equation. The negative effects of hostility and chronic experience and overt expression of anger emerged as stronger than any positive association between hardiness and healthy behaviours. Implications for nursing include anger recognition and constructive management as areas of education and support. These interventions should assist women in managing their health better and will provide fruitful areas for further study.

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A new algorithm has been developed which permits, for the first time, real-time data reduction of nadir measurements taken with a gas-filter correlation radiometer to determine tropospheric carbon-monoxide concentrations. The algorithm significantly reduces the complexity of the equations to be solved while providing accuracy comparable to line-by-line calculations. The method is based on a regression analysis technique using a truncated power series representation of the primary instrument output signals to infer directly a weighted average of trace gas concentration. The results produced by a microcomputer-based implementation of this technique are compared with those produced by the more rigorous line-by-line methods. This algorithm has been used in the reduction of MAPS Shuttle and aircraft data.

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During the second flight of the space shuttle, the measurement of air pollution from satellites (MAPS) experiment in the OSTA-1 payload acquired approximately 35 hours of radiometric measurements of the carbon monoxide mixing ratio in the middle troposphere, upper troposphere, and lower stratosphere. A gas filter radiometer operating in the 4.67-micrometer band was used to acquire the data over the region from 38 degrees N to 38 degrees S during both daytime and nighttime. The performance of the measurement system was excellent. The data reduced to date indicate the presence of significant gradients in the middle tropospheric carbon monoxide mixing ratio with both latitude and longitude over the North Atlantic, the Mediterranean Sea, and the Middle East. On the basis of comparisons with directly measured values, the accuracy of the measurements is approximately 15 percent. Comparisons of data taken on successive orbits over the same geographic region indicate that the repeatability of the measurements is approximately 5 percent.

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The major flavonoids of Pisum are derivatives of kaempferol and quercetin, including both triglucosides and acylated triglucosides in which the acyl group is p-coumaric acid. Although hydroxycinnamic acid esters of flavonoids are common pigments in many plants, neither the enzymes nor the precursors involved in their biosynthesis have been demonstrated. We report here that crude enzyme preparations extracted from peas catalyze the transfer of the p-coumaroyl moiety of p-coumaroyl:Coenzyme A to kaempferol-3-triglucoside forming kaempferol-3-(p-coumaroyl triglucoside) as the acylated product. The reaction product has been vigorously shown to be identical to the naturally occurring kaempferol-3-(p-coumaroyl triglucoside) in both chromatographic and chemical properties. The enzymatic formation of the acylated derivative occurred only minimally when incubated with the cofactors required for carboxyl group activation (ligase) and maximally when incubated with p-coumaroyl : Coenzyme A as the acyl donor.

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